EP4259818A1 - Systèmes et procédés pour détecter des biomarqueurs de la polyarthrite rhumatoïde - Google Patents

Systèmes et procédés pour détecter des biomarqueurs de la polyarthrite rhumatoïde

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Publication number
EP4259818A1
EP4259818A1 EP21904515.0A EP21904515A EP4259818A1 EP 4259818 A1 EP4259818 A1 EP 4259818A1 EP 21904515 A EP21904515 A EP 21904515A EP 4259818 A1 EP4259818 A1 EP 4259818A1
Authority
EP
European Patent Office
Prior art keywords
immunocomplex
rheumatic disease
subject
lateral flow
hsa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21904515.0A
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German (de)
English (en)
Inventor
Srinivasa R. Nagalla
Charles T. Roberts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diabetomics Inc
Original Assignee
Diabetomics Inc
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Filing date
Publication date
Application filed by Diabetomics Inc filed Critical Diabetomics Inc
Publication of EP4259818A1 publication Critical patent/EP4259818A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/18—Post-translational modifications [PTMs] in chemical analysis of biological material citrullination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • Embodiments herein relate to the field of biomarker detection, and more specifically, to systems and methods of biomarker detection for assessment of rheumatic disease.
  • RA Rheumatoid arthritis
  • DMARDs disease-modifying antirheumatic drugs
  • DMARDs may be non-biologic or biologic agents, and biologic DMARDs are usually most effective when paired with a non-biologic DMARD.
  • DMARDs can slow the progression of RA and at least partially save the joints or other tissues from permanent damage.
  • Other relevant treatments include medications (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), steroids, etc.) and physical therapy. If such treatments fail to prevent or slow joint damage, RA surgery may be performed, however surgeries come with risks such as bleeding, infection, and pain, and it is thus desirable to avoid surgery wherever possible.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • FIG. 1 illustrates a schematic diagram showing an example of a lateral flow assay for detection of anti-citrullinated human serum albumin (HSA) autoantibody-citrullinated HSA complexes in clinical samples.
  • HSA human serum albumin
  • FIG. 2A depicts a lateral flow test device that may be used in accordance with various embodiments disclosed herein.
  • FIG. 2B depicts an illustration of an example reader device for quantification of test line intensity corresponding to lateral flow test devices of the present disclosure.
  • FIG. 3 depicts another lateral flow test device that may be used in accordance with various embodiments disclosed herein.
  • FIG. 4 illustratively depicts example results of a lateral flow test device such as that depicted at FIG. 2 A.
  • FIG. 5 illustratively depicts example results of a lateral flow test device such as that depicted at FIG. 3.
  • FIG. 6 depicts a Venn diagram for anti-citrullinated albumin antibodies (ACA) positive subjects and anti-cyclic citrullinated peptide (ACCP) positive subjects in a clinical study for rheumatoid arthritis.
  • ACA anti-citrullinated albumin antibodies
  • ACCP anti-cyclic citrullinated peptide
  • RF rheumatoid factor
  • ACPAs anti-citrullinated peptide autoantibodies
  • HSA human serum albumin
  • ACAs anti-citrullinated human serum albumin autoantibodies
  • RF measure the amount of RF in blood, and can be measured in at least one of two ways, for example 1) agglutination tests and 2) nephelometry tests.
  • a method includes mixing a subject’s blood with small rubber beads (e.g., latex) that are covered with human antibodies.
  • a method includes mixing a subject’s blood with a sheep’s red blood cells that have been covered with rabbit antibodies. If RF is present, the red blood cells clump together.
  • a subject In a nephelometry test, a subject’s blood is mixed with antibodies that cause the blood to clump if RF is present. Detection may be via laser- light. Specifically, the test monitors for an amount of light being blocked by the blood sample when laser light is shone onto the sample. Higher levels of RF are associated with greater clumping and a cloudier sample that results in less light passing through a tube that comprises the sample. Other tests for RF have been developed, including enzyme-linked immunosorbent assays (EEISAs) and radioimmune assays (RIAs).
  • EEISAs enzyme-linked immunosorbent assays
  • RIAs radioimmune assays
  • ACPA assays comprise kits employing a substrate derived from a synthetic cyclic citrullinated peptide, but differ in incubation time, volume and dilution of serum, type of conjugate and of enzymatic substrate, and range of units reported and thresholds for positive results.
  • Newer assays detect non-cyclic citrullinated peptides.
  • Such assays are, in general, based on detection of autoantibodies by EEISA, microparticle enzyme immunoassay (MEIA), or immunoenzyme fluorimetry.
  • embodiments herein provide devices and methodology for detecting one or more biomarkers of RA, and managing RA based at least in part on levels of the one or more biomarkers of RA detected in a biological sample using the methods and devices disclosed herein.
  • a method of detecting anti-citrullinated human serum albumin (HSA) autoantibody in a biological sample comprises obtaining the biological sample from a subject, and applying the biological sample to a lateral flow device under conditions sufficient for formation of an immunocomplex comprising anti-HSA antibody coupled to citrullinated HSA that is in turn coupled to anti-citrullinated HSA autoantibody.
  • the method further includes detecting a quantity of the immunocomplex, and managing rheumatic disease associated with the subject.
  • the biological sample is serum or plasma.
  • the anti-HSA may be coupled to a detectable label, for example a colloidal gold particle although other detectable labels are within the scope of this disclosure as discussed infra.
  • detecting the quantity of the immunocomplex may be via a reader configured with an image analysis system. Detecting the quantity of the immunocomplex may further comprise quantifying relative amounts of the immunocomplex to a control complex.
  • control complex may comprise streptavidin coupled to a detectable label.
  • the detectable label may be a colloidal gold particle, although other detectable labels are within the scope of this disclosure.
  • streptavidin coupled to its detectable label may be captured via the lateral flow device by biotin-coupled to a protein (e.g., bovine serum albumin (BSA)).
  • a protein e.g., bovine serum albumin (BSA)
  • the immunocomplex may be captured via the lateral flow device by an antibody cocktail that includes anti-human IgA, IgG and IgM antibodies.
  • managing rheumatic disease in the subject may be a function of the quantity of the immunocomplex detected.
  • managing rheumatic disease in the subject may include selecting one or more therapeutic agents appropriate to manage rheumatic disease based on the quantity of the immunocomplex detected, and the selecting may include determining an effective amount of the one or more therapeutic agents to appropriately manage rheumatic disease.
  • the one or more therapeutic agents may be selected from aspirin, nonsteroidal anti-inflammatory agents, corticosteroids, disease-modifying antirheumatic drugs (DMARDs), penicillamine, Rituximab (monoclonal anti-CD20 antibody), cyclosporine, Infliximab (monoclonal anti tumor necrosis factor antibody), Leflunomide (pyrimidine synthesis inhibitor), Anakinra (recombinant human interleukin- 1 receptor antagonist), Etanercept (protein therapy based on a binding fragment of the tumor necrosis factor alpha receptor), and Adalimumab (monoclonal anti tumor necrosis antibody).
  • managing rheumatic disease may include recommending to the subject one or more antirheumatic lifestyle modifications. Furthermore, in some additional or alternative examples, managing rheumatic disease may include performing the method of detecting citrullinated human serum albumin again at a later time, and adjusting the managing of rheumatic disease as a function of the quantity of the immunocomplex detected at the later time. Such a cycle can be repeated any number of times to manage rheumatic disease in the subject.
  • managing rheumatic disease reduces at least one sign or symptom associated with rheumatic disease.
  • the rheumatic disease may be RA.
  • Another embodiment includes a method of detecting a first biomarker and at least a second biomarker associated with rheumatic disease in a biological sample of a subject.
  • the method may comprise obtaining the biological sample from the subject, and applying the biological sample to a lateral flow device under conditions sufficient for formation of a first immunocomplex comprising at least the first biomarker and a first labeled biomolecule capable of recognizing the first biomarker, and a second immunocomplex comprising at least the second biomarker and a second labeled biomolecule capable of recognizing the second biomarker.
  • the first biomarker is citrullinated HSA.
  • the second biomarker is one of rheumatoid factor (RF) and anti-citrullinated peptide antibody (ACAP).
  • the method may further comprise detecting a quantity of each of the first immunocomplex and the second immunocomplex, and managing rheumatic disease associated with the subject.
  • the method may further comprise, via the lateral flow device, detecting a third biomarker that is the other of RF or ACAP by applying the biological sample to the lateral flow device under conditions additionally sufficient for formation of a third immunocomplex comprising at least the third biomarker and a third labeled biomolecule capable of recognizing the third biomarker.
  • detecting the quantity of each of the first immunocomplex and the second immunocomplex further comprises additionally detecting a quantity of the third immunocomplex, and managing rheumatic disease associated with the subject.
  • managing rheumatic disease in the subject may be a function of a relative quantity of at least two of each of the first immunocomplex, the second immunocomplex and/or the third immunocomplex with respect to one another.
  • managing rheumatic disease in the subject may be a function of a relative quantity of each of the first immunocomplex, the second immunocomplex and the third immunocomplex with respect to one another.
  • managing rheumatic disease in the subject may further comprise selecting one or more therapeutic agents appropriate to manage rheumatic disease, and the selecting may include determining an effective amount of the one or more therapeutic agents in order to appropriately mange rheumatic disease.
  • the one or more therapeutic agents may be selected from aspirin, nonsteroidal anti-inflammatory agents, corticosteroids, disease-modifying antirheumatic drugs (DMARDs), penicillamine, Rituximab (monoclonal anti-CD20 antibody), cyclosporine, Infliximab (monoclonal anti tumor necrosis factor antibody), Leflunomide (pyrimidine synthesis inhibitor), Anakinra (recombinant human interleukin- 1 receptor antagonist), Etanercept (protein therapy based on a binding fragment of the tumor necrosis factor alpha receptor), and Adalimumab (monoclonal anti tumor necrosis antibody).
  • managing rheumatic disease may further comprise recommending to the subject one or more antirheumatic lifestyle modifications.
  • managing rheumatic disease may further comprise performing the method of detecting the first biomarker and at least the second biomarker again at a later time, and adjusting the managing of rheumatic disease as a function of the quantity of at least two of the first immunocomplex, the second immunocomplex, and/or the third immunocomplex detected at the later time.
  • the test line may include one or more immobilized biomolecules capable of recognizing the anti-citrullinated HSA autoantibodies
  • the control line may include a second control biomolecule capable of binding (e.g., recognizing) the first control molecule.
  • the first detectable label and the second detectable label may be the same. In other examples, the first detectable label and the second detectable label may be different.
  • the first detectable label and the second detectable label may be selected from fluorescent tags/particles, enzymatic linkages, radioactive isotopes, microspheres, and nanoparticles.
  • the one or more immobilized biomolecules capable of recognizing anti- citrullinated HSA autoantibodies may comprise an antibody cocktail that includes anti-human IgA, IgM and IgG.
  • the first control biomolecule may be streptavidin.
  • the second control biomolecule may be biotin coupled to a protein (e.g., bovine serum albumin (BSA)).
  • BSA bovine serum albumin
  • the lateral flow device detects RA in subjects with a sensitivity of about 39% and a specificity of about 99.8%.
  • Coupled may mean that two or more elements are in direct physical or electrical contact. However, “coupled” may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other.
  • a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B).
  • a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).
  • a phrase in the form “(A)B” means (B) or (AB) that is, A is an optional element.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects, for example dogs, cats, mice, etc.
  • Antibody A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of a protein discussed herein (which can include another antibody), or a fragment of any of these proteins.
  • the term “specifically binds” refers to, with respect to an antigen such as the proteins/peptides discussed herein, the preferential association of an antibody or other ligand, in whole or part, with the protein.
  • a specific binding agent binds substantially only to a defined target, such as protein of interest.
  • a citrullinated peptide or protein- specific binding agent is an agent that binds substantially to a citrullinated protein or peptide. If an agent, such as an antibody, specifically binds a particular citrullinated peptide or protein, it does not specifically bind other peptides or proteins that are non-citrullinated. A minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Specific binding can be distinguished as mediated through specific recognition of the antigen.
  • a variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • Antibodies can include a heavy chain and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • VH region and VL region are responsible for binding the antigen recognized by the antibody.
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies), etc. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
  • a “monoclonal antibody” is an antibody produced by a single clone of B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed “hybridomas.”
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • Anti-citrullinated human serum albumin autoantibodies Refers to autoantibodies that recognize citrullinated human serum albumin (HSA). ACAs comprise a subset of anti-citrullinated protein antibodies (ACPAs).
  • ACPAs Anti-citrullinated peptide autoantibody
  • IgG Autoantibodies with different isotypes usage (e.g., IgG, IgA, IgM) that recognize the nonessential amino acid citrulline in peptides and/or proteins.
  • Citrulline is formed as a result of post-translational modification of arginine, catalyzed by intracellular enzymes known as peptidylarginine deiminases (PADs).
  • PADs peptidylarginine deiminases
  • the generation of ACPAs appears to be a specific phenomenon for RA and can start years before the onset of disease. Discussed herein, ACPAs include but are not limited to anti-cyclic citrullinated peptide antibodies (anti-CCP).
  • Autoantibody Antibodies that react with self-antigens. These antigens may be found in a wide variety of cell types in some examples, be highly specific for particular cell types in other examples, or may comprise circulating antigens throughout a body of an animal. Autoantibodies are formed in an animal’s own body, and are capable in examples of directly destroying cells that have the self-antigen on them or can render them more susceptible to white blood cells that in turn can destroy them. Some autoimmune diseases can be caused by autoantibodies.
  • Autoimmune disease A disease in which an animal’s body attacks parts of its body, including but not limited to joints and skin, as foreign.
  • autoimmune disease include but are not limited to Rheumatoid arthritis (RA), Psoriasis/psoriatic arthritis, Multiple sclerosis, Type 1 diabetes, Systemic lupus erythematosus (SLE), Inflammatory bowel disease (IBD), Autoimmune vasculitis, Celiac disease, Hashimoto’s thyroiditis, Graves disease, Sjogren’s syndrome, Pernicious anemia, Myasthenia gravis, Vasculitis, Ankylosing spondylitis, among others.
  • RA Rheumatoid arthritis
  • Psoriasis/psoriatic arthritis Multiple sclerosis
  • Type 1 diabetes Type 1 diabetes
  • SLE Systemic lupus erythematosus
  • IBD Inflammatory bowel disease
  • Autoimmune vasculitis Celiac disease, Hashimoto’s
  • Antirheumatic agent refers to agents used in the therapy of inflammatory arthritis, for example Rheumatoid arthritis.
  • An anti-rheumatic agent can also be used in the therapy of psoriatic arthritis, ankylosing spondylitis, idiopathic juvenile arthritis, among others.
  • antirheumatic agents include but are not limited to aspirin, nonsteroidal anti-inflammatory agents, corticosteroids, DMARDs, penicillamine, Rituximab (monoclonal anti-CD20 antibody), cyclosporine, Infliximab (monoclonal anti tumor necrosis factor antibody), Leflunomide (pyrimidine synthesis inhibitor), Anakinra (recombinant human interleukin- 1 receptor antagonist), Etanercept (protein therapy based on a binding fragment of the tumor necrosis factor alpha receptor), Adalimumab (monoclonal anti tumor necrosis antibody), and the like.
  • Antirheumatic lifestyle modifications Changes to lifestyle, habits and practices intended to alleviate the symptoms of rheumatoid arthritis. Examples include changes to physical activity (e.g., increase in exercise), changes that lead to a reduction in body mass index (BMI), reduction or cessation of smoking, increase in vitamin and/or mineral intake (e.g., increased vitamin D intake), reduction in stress levels, increased quality of sleep, and the like.
  • Binding A specific interaction between two or more molecules, such as the binding of an antibody and an antigen (for example an antibody to an antigen). In one embodiment, specific binding is identified by a dissociation constant (Kd). In another embodiment, binding affinity is measured by a dissociation rate. In yet another embodiment, a binding affinity is measured by a competition assay.
  • Contacting includes in solution and solid phase, for example contacting a blood or serum protein with a test agent.
  • contacting includes contacting a sample with an antibody, for example contacting a sample that contains a protein of interest.
  • contacting includes contacting a sample to a sample well/pad of a lateral flow test strip.
  • Citrullinated Peptides or proteins which have one or more amino acids that have been deimidated, for example peptides or proteins in which one or more arginine residues have been deimidated to citrulline.
  • Effective amount An amount of therapeutic agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. When administered to a subject, a dosage will generally be used that will achieve target tissue/cell concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and/or underlying causes of any of a disorder or disease. In a representative example, an “effective amount” is a therapeutically effective amount in which the agent alone or with one or more additional therapeutic agent(s) induces the desired response such as reduction in one or more symptoms associated with RA.
  • an effective amount is an amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents (e.g., methotrexate), induces the desired response.
  • a pharmaceutically acceptable carrier or one or more additional therapeutic agents e.g., methotrexate
  • a desired response is to increase the subject's survival time and/or improve the subject’s quality of life, for example by reducing a number and/or amount of signs/symptoms associated with RA.
  • a desired response is to increase the subject's survival time and/or improve the subject’s quality of life by slowing or eliminating progression of disease, such as slowing or eliminating the progression of RA.
  • a pharmaceutical preparation may decrease the progression of the disease, syndrome, etc., by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the progression typical in the absence of the pharmaceutical preparation.
  • treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently.
  • Effective amounts of the agents described herein can be determined in many different ways, such as, for example, assaying for a reduction in of one or more signs or symptoms associated with RA in the subject or measuring the expression level of one or more biological molecules (e.g., RF, ACAP, ACA) known to be associated with RA. Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.
  • assaying for a reduction in of one or more signs or symptoms associated with RA in the subject or measuring the expression level of one or more biological molecules (e.g., RF, ACAP, ACA) known to be associated with RA.
  • Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.
  • the disclosed therapeutic agents can be administered in a single dose, or in several doses, for example hourly, daily, weekly, monthly, yearly, during a course of treatment.
  • the effective amount can be dependent on the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • one or more of dosage, frequency of administration, type of agent, etc. can be adjusted depending on changes in signs or symptoms, changes in biomarker levels, etc., associated with RA.
  • Immunoassay A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. The presence of antigen and in some examples the amount of antigen present, can be measured.
  • Inflammation refers to a condition that occurs in animals when tissues are injured in some way, causing the damaged cells to release particular chemicals including but not limited to histamine, bradykinin, prostaglandins, and the like.
  • Tissue injury may be caused by bacteria, trauma, toxins, heat, diet, repetitive use, stationary lifestyle, and the like.
  • Inflammation may be acute or chronic. Acute inflammation can last anywhere from 1-2 days to 2-6 weeks. Chronic inflammation can continue for months or even years and can have links to various diseases including but not limited to rheumatoid arthritis.
  • inflammation is used interchangeably with inflammatory response.
  • Lateral flow assay A platform for the detection and quantification of analytes in complex mixtures, including but not limited to urine, saliva, sweat, serum, plasma, whole blood, and the like. Lateral flow assays can be categorized into different types, such as lateral flow immunoassays (LFIAs) in which antibodies are exclusively used as recognition elements, and nucleic acid LFAs, used for the detection of amplicons, which can be formed during a polymerase chain reaction (PCR).
  • LFIAs lateral flow immunoassays
  • PCR polymerase chain reaction
  • the principle of LFAs relies on a liquid sample or its extract containing an analyte of interest moving without the assistance of external forces through a number of zones of a lateral flow test strip, on which molecules that can interact with the analyte are attached.
  • Such interaction molecules can be labeled with a detectable label to facilitate detection of the interaction.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or other protein/peptide, to facilitate detection of that molecule or a molecule (or complex) to which the labeled molecule is bound.
  • Specific, non-limiting examples of labels include fluorescent tags/particles, enzymatic linkages, radioactive isotopes (for example 14C, 32P, 1251, 3H isotopes and the like), microspheres, nanoparticles, and the like.
  • nanoparticles may comprise one or more of metal nanoparticles, magnetic nanoparticles, silica nanoparticles, and latex nanoparticles.
  • the fluorescent particles can include one or more of Alexa fluoro 350, 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, ruby (tris (2,2-bipyridyl) ruthenium ), FITC (fluoresein isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6- carboxy-tetramethyl-rhodamine), Texas Red, DAPI (4,6-diamidino -2-phenylindole, and coumarin, or may be time-resolved fluorescence (TRF).
  • Alexa fluoro 350 405, 430, 488, 500, 514, 633, 647, 660, 680, 700, cy3, cy5, cy7, ruby (tris (2,2-bipyridyl) ruthenium ), FITC (fluoresein isothio
  • Patient includes human and non-human animals.
  • the preferred patient for treatment is a human.
  • Patient and subject are used interchangeably herein.
  • Pharmaceutical agent A chemical compound or composition capable of inducing a desired therapeutic (including in some examples a prophylactic effect) when properly administered to a subject.
  • the pharmaceutically acceptable salts of the compounds of this invention include, but are not limited to, those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.
  • salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof.
  • This term refers to pharmaceutical agents, pharmaceutical compositions, and drugs acceptable for both human and veterinary uses.
  • Proteome A significant portion of proteins in a biological sample at a given time.
  • a “proteomic profile” is a representation of the expression pattern of a plurality of proteins in a biological sample, such as plasma or serum, at a given time.
  • Rheumatoid arthritis A disease that causes joint inflammation, pain and underlying bone loss, and occurs when the body’s immune system attacks lining of the joints. In severe cases, internal organs can be attacked. Signs/symptoms of RA include but are not limited to joint stiffness, loss of energy, low grade fever, loss of appetite, dry eyes and/or mouth, firm lumps referred to as rheumatoid nodules that grow beneath the skin in places such as the elbows and hands, among others.
  • Rheumatoid factor Refers to antibodies directed against the Fc (fragment crystallizable region) fragment of immunoglobulin G (IgG) .
  • the antibodies referred to as RFs are heterogeneous and generally are comprised of immunoglobulin M (IgM).
  • RFs are used as a marker in individuals with suspected RA or other autoimmune conditions.
  • Sample biological sample: A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, blood (serum and/or plasma), urine, saliva, milk, and the like. In one example, the sample includes serum and/or plasma from a subject suspected of being afflicted with RA.
  • Signs or symptoms Any subjective evidence of disease or of a subject's condition, e.g., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state.
  • a “sign” is any abnormality indicative of disease, discoverable on examination or assessment of a subject.
  • a sign is generally an objective indication of disease. Signs include, but are not limited to any measurable parameters such as tests for detecting RA, including antibody blood tests, for example, tests for one or more of RF, ACPAs, ACA, antinuclear antibodies (ANA), and the like.
  • Streptavidin A protein with an extraordinarily strong affinity for biotin (also known as vitamin B7 or vitamin H). Streptavidin as discussed herein may comprise an oligomer of streptavidin, streptavidin mutein or analog, avidin, an avidin mutein or analog (such as neutravidin) or a mixture thereof. In general, streptavidin naturally occurs as a tetramer of four identical subunits, i.e. it is a homo-tetramer, where each subunit contains a single binding site for biotin, a biotin derivative or analog or a biotin mimic.
  • Subject A term that includes both human and veterinary individuals, for example mammals, such as humans.
  • Therapeutic agent A substance that demonstrates some therapeutic effect by restoring or maintaining health, such as by alleviating one or more signs or symptoms associated with a disease or physiological disorder, or delaying (including preventing) progression or onset of a disease.
  • the therapeutic agent is a chemical or pharmaceutical agent, or a prodrug.
  • a therapeutic agent may be an antirheumatic agent.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount or dose sufficient to inhibit or prevent onset or advancement, to treat outward symptoms, or to cause regression, of a disease.
  • the therapeutically effective amount or dose also can be considered as that amount or dose capable of relieving symptoms caused by the disease.
  • a therapeutically effective amount or dose of an antirheumatic agent is that amount or dose sufficient to achieve a stated therapeutic effect.
  • a therapeutically effective amount of an antirheumatic agent is an amount that reduces the signs of, symptoms of, or laboratory findings associated with RA; delays the progression of RA; or lowers one or more of biomarker level(s) associated with RA, inflammation associated with RA, and the like.
  • Treating a disease A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition including but not limited to a RA, such as a sign or symptom of RA. Treatment can induce remission or cure of a condition or slow progression, for example. In some instances, treating a disease can include inhibiting the full development of a disease, for example preventing development adverse conditions associated with RA. Prevention of a disease does not require a total absence of disease.
  • treating a disease can be a reduction in severity of some or all clinical symptoms of the disease or condition, a reduction in the number of relapses of the disease or condition, an improvement in the overall health or well- being of the subject, decrease in other parameters well known in the art that are specific to the particular disease or condition, and combinations of such factors. Discussed herein, “treating” a disease is interchangeable with “managing” a disease, such as RA.
  • anti-citrullinated HSA autoantibody levels may be assessed using a lateral flow device.
  • Various lateral flow assay methods may be utilized to test for the presence or absence or quantity of an analyte, such as anti-citrullinated HSA autoantibody bound to citrullinated HSA, in a biological sample.
  • an analyte such as anti-citrullinated HSA autoantibody bound to citrullinated HSA
  • a “sandwich” assay method of the present disclosure may rely on one or more antibodies (or in some examples other recombinant proteins) immobilized on a solid support, which forms part of a complex that includes a labeled antibody or other labeled biomolecule.
  • the label may be colloid gold particles, however other labels are within the scope of this disclosure. Examples include but are not limited to enzymes, fluorescently-labeled microspheres, colored microspheres, and the like, provided the label enables detection and/or quantification of analyte bound to a test line.
  • lateral flow test strips of the present disclosure comprise a solid support on which sample receiving area (e.g., sample pad) and target capture zones (e.g., capture pad) are supported.
  • the solid support material is one which is capable of supporting the sample receiving area and target capture zones and providing for the capillary flow of sample out from the sample receiving area to the target capture zones when the lateral flow test strip is exposed to an appropriate solvent or buffer which acts as a carrier liquid for the sample.
  • Specific classes of materials that may be used as support include organic or inorganic polymers, and natural and synthetic polymers. More specific examples of suitable solid supports include, without limitation, glass fiber, cellulose, nylon, crosslinked dextran, various chromatographic papers and nitrocellulose. One particularly useful material is nitrocellulose.
  • FIG. 1 depicted is a schematic diagram showing an example of a lateral flow immunoassay 100 that may be used in accordance with various embodiments disclosed herein.
  • an antibody cocktail 101 comprising one or more of anti-human IgA, IgM and IgG is immobilized on a test line (not specifically shown at FIG. 1), and biotin-coupled bovine serum albumin (BSA) 102 is immobilized on a control line (not specifically shown at FIG. 1), of a membrane 103 (e.g., nitrocellulose membrane).
  • BSA bovine serum albumin
  • An anti-HSA antibody 104 is coupled to a label 105 (e.g. colloidal gold particles) that serves as a test detector.
  • label 105 e.g. colloidal gold particles
  • the anti-HSA antibody 104 binds to, and hence detects, anti-citrullinated HSA autoantibody 113 bound to citrullinated HSA 114 that has been captured by the antibody cocktail 101.
  • Streptavidin 106 coupled to another label 107 serves as the control detector by binding to the biotin 109 coupled to BSA 110 on the control line.
  • FIG. 2A depicts an example lateral flow test strip that may be used in accordance with various embodiments herein disclosed.
  • the lateral flow test strip includes at least sample well 201 (also referred to herein as a sample pad), conjugate pad 202, capture pad 206 which includes test line 203 and control line 204, and optionally, absorption pad 205.
  • sample well 201 also referred to herein as a sample pad
  • conjugate pad 202 also referred to herein as a sample pad
  • capture pad 206 which includes test line 203 and control line 204
  • absorption pad 205 optionally, absorption pad 205.
  • labeled anti-HSA antibody e.g., anti-HSA antibody 104 at FIG. 1
  • labeled streptavidin e.g., streptavidin 106 at FIG.
  • the conjugate pad may be treated with a solution that may include but is not limited to sodium borate, dextran, BSA, Tween-20, sodium azide, and the like.
  • a solution may be in a pH range of 7.5-8.5, for example pH 8.0.
  • the drying of the labeled anti-HSA antibody and the labeled streptavidin may be conducted at temperatures ranging from 40-60°C, for example 50°C.
  • a biological sample for example blood (e.g., serum or plasma), saliva, urine, milk, and the like, may in some examples be diluted in an appropriate buffer (e.g., running buffer comprising one or more of HEPES, CaC12, NaCl, NaN3, Tween-20 and 0.01% polyvinyl alcohol), and applied to sample pad 201.
  • an appropriate buffer e.g., running buffer comprising one or more of HEPES, CaC12, NaCl, NaN3, Tween-20 and 0.01% polyvinyl alcohol
  • capillary action allows the components of the sample to hydrate and (potentially) interact with labeled anti-HSA antibody, as well as to hydrate the labeled streptavidin, at the conjugate pad 202.
  • an appropriate buffer e.g., running buffer comprising one or more of HEPES, CaC12, NaCl, NaN3, Tween-20 and 0.01% polyvinyl alcohol
  • labeled-anti- HSA antibody binds to citrullinated HSA- anti-citrullinated HSA autoantibody complex
  • the formed immunocomplex may then further migrate to test line 203, where the complex may be captured by the immobilized antibody cocktail, via the antibody cocktail binding to anti- citrullinated HSA autoantibodies.
  • the labeled streptavidin may migrate past the test line to the control line, to be captured by the biotin (e.g., biotin 109 at FIG. 1) coupled to BSA (e.g., BSA 110).
  • the lateral flow test strip 200 may be scanned via a reader device 250.
  • Reader 250 may utilize image analysis for quantification of relative amounts of labeled anti-HSA antibody at the test line and labeled streptavidin at the control line.
  • reader 250 may be configured to quantify intensity of a particular color (e.g., red color reflected by the colloidal gold particles) corresponding to the control line and the test line.
  • reader 250 may comprise an image analysis system 251.
  • Image analysis system 251 may include but is not limited to one or more of a camera (e.g., CCD camera), lens (e.g., micro lens), light source (e.g., white light-emitting diode (LED) source), a light controller (e.g., LED controller) and a computing device (e.g., computer).
  • Software programs stored on a memory of the computer may be used to analyze images acquired by image analysis system 251.
  • a software program or programs may be used to measure maximum peak area values of the test line and control line within a detection area of the lateral flow test strip.
  • peak area may be calculated by adding intensity values under an intensity profile of the test line or control line.
  • Intensity associated with the control line may be used as a normalizing factor, such that experimental results may be expressed as the peak area of the test line divided by the peak area of the control line.
  • the description as to how the image analysis system 251 operates is meant to be illustrative and non-limiting in nature.
  • reader 250 may include a display 252 that enables a user to interact with the reader 250 and corresponding image analysis system 251, to control one or more parameters for acquiring and/or processing images.
  • image reader 250 may include a recognition module for test and lot-specific calibration and quantification of each lateral test strip.
  • the recognition module may be capable of recognizing/decoding information stored in a quick response (QR) code, attached to each lateral flow test strip.
  • the lateral flow immunoassay depicted at FIG. 1 is advantageous in that the detection methodology does not necessitate the generation of anti-citrullinated HSA antibodies, but instead relies on anti-HSA antibodies that are readily available and can be used across a wide sample population. Specifically, it is herein recognized that in no case may subjects have circulating anti-native HSA autoantibodies, as autoantibodies are just generated within subjects to anti-citrullinated HSA and not non-citrullinated HSA. Thus, in no case will the lateral flow assay of FIG.
  • the lateral flow assay depicted at FIG. 1 can detect is anti-citrullinated HSA in complex with anti-citrullinated HSA autoantibodies, even though the detection antibody is labeled anti-HSA and not specifically labeled anti-citrullinated HSA.
  • a lateral flow device there may be second, or third or even fourth (or more) test lines. It is herein recognized that this type of lateral flow device may enable determination of relative quantities of at least two of RF, ACPAs, and ACAs, in an example. Determination of relative quantities of at least two of RF, ACPAs, and ACAs may improve an ability to diagnose and/or manage RA.
  • the presence of more than one biomarker of rheumatic disease (e.g., RA) and the relative quantities of such biomarkers in relation to each other may be indicative of an increased risk and/or more severe progression of rheumatic disease (e.g., RA), which in turn may be relied upon for management of the rheumatic disease.
  • FIG. 3 an example of a multi-test line lateral flow device 300, is shown.
  • three different test lines comprising first test line 303, second test line 304, and third test line 305, are included.
  • control line 306 sample pad 301, conjugate pad 302 and absorption pad 307.
  • the operation of this particular embodiment is similar to that described above with regard to FIGS.
  • a second detector reagent e.g., another antibody conjugated to a detectable label
  • the second test line 304 may include a second specific binding partner having affinity for a second analyte (e.g., ACPA).
  • a third detector reagent e.g., another antibody conjugated to a detectable label
  • the third test line 305 may include a third specific binding partner having affinity for the third analyte (e.g., RF).
  • the first test line 303 may comprise the antibody cocktail (e.g., antibody cocktail 101) immobilized on the test line for capture of anti-citrullinated HSA antibody (e.g., 113) bound to citrullinated HSA (e.g., 114) as discussed above with regard to FIG. 1.
  • the first test line may comprise citrullinated HSA.
  • the first, second, and third detector reagents may be the same, whereas in other embodiments one or more of the first, second, and/or third detector reagents may be different.
  • the first test line may comprise a test line for ACA
  • the second test line may comprise a test line for ACPA
  • the third test line may comprise a test line for RF.
  • the ACPAs may recognize mutated and citrullinated vimentin (MCV), and this aspect may be used in the lateral flow detection scheme.
  • MCV citrullinated vimentin
  • one or more citrullinated peptides may be recognized by ACPAs, and this aspect may be used in the lateral flow detection scheme.
  • RF may be recognized via a purified Fc-part of human immunoglobulin, and this aspect may be used in the lateral flow detection scheme. In this way, relative amounts of RF, ACPAs and ACAs may be determined on a single lateral flow device.
  • lateral flow devices of the present disclosure may be prepared differently than the above example that includes three test lines.
  • a lateral flow device may detect just one biomarker for RA, or just two biomarkers for RA.
  • a lateral flow device of the present disclosure may be configured to just detect ACA, via the methodology discussed with regard to FIG. 1 and not be additionally configured to test other RA biomarkers (e.g., RF and/or ACPA).
  • a lateral flow device may be configured to detect just RF and ACPA.
  • purified recombinant MCV and purified Fc-part of human immunoglobulin may be immobilized at each of the second test line and third test line, respectively.
  • a detector label e.g., colloidal gold particles
  • Other examples are within the scope of this disclosure.
  • a lateral flow device may be configured to detect ACA and RF but not ACPA.
  • a lateral flow device may be configured to detect ACA and ACPA but not RF.
  • control line e.g., control line 306
  • the control line may be configured in similar fashion as that discussed with regard to FIG. 1 (e.g., striped with biotin-conjugated BSA), for detection via labeled streptavidin, however other alternatives for controls are within the scope of this disclosure.
  • the control line may be striped with anti-goat IgG, or protein L. Other examples are within the scope of this disclosure.
  • such a lateral flow device may be configured as follows. Specifically, the first test line (e.g., test line 303 at FIG. 3 may be configured to detect ACA, the second test line (e.g., test line 304 at FIG. 3) may be configured to detect ACPA, and the third test line (e.g., test line 305 at FIG. 3) may be configured to detect RF.
  • the first test line e.g., test line 303 at FIG. 3
  • the second test line e.g., test line 304 at FIG. 3
  • the third test line e.g., test line 305 at FIG. 3
  • the first test line may be striped with (e.g., may comprise) citrullinated HSA
  • the second test line may be striped with a citrullinated peptide mixture including or excluding MCV
  • the third test line may be striped with the purified Fc-part of human immunoglobulin.
  • One or more of the citrullinated HSA, citrullinated peptide mixture, and/or Fc-part of human immunoglobulin may be recombinantly produced and purified for striping of the corresponding test lines of the lateral flow device.
  • the control line may comprise one or more of anti-goat IgG, protein L, and the like.
  • the detector may comprise a goat-anti-human IgG, IgM and IgA coupled to a detectable label.
  • the detectable label may comprise one or more of fluorescent tags/particles, enzymatic linkages, radioactive isotopes, microspheres, and nanoparticles.
  • the detectable is colloidal gold particle(s). In this way, at least two (e.g., 3) RA biomarkers may be detectable on a single lateral flow device.
  • Sample pads as discussed are components of lateral flow devices of the present disclosure.
  • the sample pads initially receive the sample (e.g., biological sample), and may serve to remove particulates from the sample.
  • a sample pad such as glass fiber, woven fibers, screen, non-woven fibers, cellulosic fibers or paper
  • a cellulose sample pad may be beneficial if a large bed volume (e.g., 250 pl/cm2) is a factor in a particular application.
  • Sample pads may be treated with one or more release agents, such as buffers, salts, proteins, detergents, and surfactants.
  • Such release agents may be useful, for example, to promote resolubilization of conjugate-pad constituents, and to block non-specific binding sites in other components of a lateral flow device, such as a nitrocellulose membrane.
  • Representative release agents include, for example, trehalose or glucose (l%-5%), PVP or PVA (0.5%-2%), Tween 20 or Triton X-100 (0.1 %- 1%), casein (1%- 2%), SDS (0.02%-5%), and PEG (0.02%-5%).
  • the conjugate pads (e.g., 202) of the present disclosure serve to, among other things, hold a detector reagent. Suitable materials for the conjugate pad include glass fiber, polyester, paper, or surface modified polypropylene.
  • a detector reagent may be applied externally, for example, from a developer bottle, in which case a lateral flow device need not contain a conjugate pad (see, for example, U.S. Pat. No. 4,740,468).
  • Detector reagent(s) contained in a conjugate pad is typically released into solution upon application of the test sample.
  • a conjugate pad may be treated with various substances to influence release of the detector reagent into solution.
  • the conjugate pad may be treated with PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%).
  • Other release agents include, without limitation, hydroxypropylmethyl cellulose, SDS, Brij 35, and 0-lactose. A mixture of two or more release agents may be used in any given application.
  • the detector reagent in conjugate pad is a gold-conjugated antibody, or a plurality of different gold-conjugated antibodies. In some examples, different antibodies serving as different detectors may be labeled with different detector labels.
  • an absorption pad e.g., 205
  • the absorbent pad acts to enable an increase in the total volume of sample that enters the device. This increased volume can be useful, for example, to wash away unbound analyte from the membrane. Any of a variety of materials is useful to prepare an absorbent pad, for example, cellulosic filters or paper.
  • an absorbent pad can be paper (i.e., cellulosic fibers).
  • One of skill in the art may select a paper absorbent pad on the basis of, for example, its thickness, compressibility, manufacturability, and uniformity of bed volume. The volume uptake of an absorbent made may be adjusted by changing the dimensions (usually the length) of an absorbent pad.
  • FIG. 4 depicted are illustrative examples of results that can be obtained from a lateral flow device that includes one test line for detection of ACA, and a control line. Shown on the left is an example of an invalid test, lacking significant signal at both the control line and the test line.
  • the middle illustration depicts a lateral flow device that shows a valid test with a negative result for ACA. Specifically, a signal is observed at the control line but not at the ACA test line, hence the biological sample used for testing is negative for ACA in the middle illustration.
  • the illustration on the right depicts a lateral flow device that shows a positive result for ACA. Specifically, a signal is observed at both the control line and the ACA test line, indicating that the biological sample used for testing is positive for ACA in the illustration on the right at FIG. 4.
  • the lateral flow devices of the present disclosure may be read by a reader (e.g., reader 250), to enable quantitation of an amount of ACA in the biological sample.
  • a reader e.g., reader 250
  • particulars of RA management may be different depending on levels of ACA detected.
  • an effective amount of a therapeutic agent e.g., antirheumatic agent
  • a subject to manage RA e.g., alleviate at least one sign or symptom of RA
  • a function of ACA amount determined in the biological sample may be different as a function of ACA amount determined in the biological sample.
  • a particular therapeutic agent or agents may be used as a function of one ACA amount (e.g., one amount range) determined in a biological sample, and a different therapeutic agent or agents may be used for another ACA amount (e.g., another amount range), to alleviate at least one sign or symptom of RA.
  • managing RA may include one or more particular antirheumatic lifestyle modifications, which may be a function of a determined amount of ACA in a biological sample. As examples, less aggressive treatment management may be used when lesser amounts of ACA are detected, and more aggressive treatment management may be used when greater amounts of ACA are detected.
  • Less aggressive treatments can include but are not limited to lower dosages of therapeutic agents (e.g., antirheumatic agents), lesser numbers (e.g., different types) of therapeutic agents provided to the subject, and less stringent behavioral lifestyle modifications (e.g., less restrictive diet, lesser emphasis on exercise, and the like), as compared to more aggressive treatments.
  • therapeutic agents e.g., antirheumatic agents
  • lesser numbers e.g., different types
  • less stringent behavioral lifestyle modifications e.g., less restrictive diet, lesser emphasis on exercise, and the like
  • FIG. 5 depicted are a series of lateral flow devices showing example results in an example where the devices are capable to detect a plurality of RA biomarkers.
  • the biomarkers detectable include ACA, RF and ACPA.
  • Device 502 illustrates an example of an invalid test, as none of the biomarkers are detected, nor is a control signal detected.
  • Device 504 illustrates an example of a valid test for which a biological sample is negative for each of RA, ACA and ACPA.
  • Device 506 illustrates an example of a valid test for which a biological sample is positive for ACA, but negative for ACPA and RF.
  • Device 508 depicts an example of a valid test for which a biological sample is positive for ACPA, but negative for ACA and RF.
  • Device 510 depicts an example of a valid test for which a biological sample is positive for RF, but negative for ACA and ACPA.
  • Device 512 depicts an example of a valid test for which a biological sample is positive for both ACA and ACPA, but negative for RF.
  • Device 514 depicts an example of a valid test for which a biological sample is positive for ACA and RF, but negative for ACPA.
  • Device 516 depicts an example of a valid test for which a biological sample is positive for ACPA and RF, but negative for ACA.
  • Device 518 depicts an example of a valid test for which a biological sample is positive for each of ACA, ACPA and RF.
  • a reader e.g., reader 250
  • a biological sample tested on a lateral flow device of the present disclosure may detect all three of RF, ACPA and ACA, with RF being three times as abundant as ACPA, which in turn may be two times as abundant as ACA.
  • RA RA ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • rheumatic disease e.g., RA
  • the lateral flow device described above with regard to at least FIG. 1 and FIG. 2A may be used for a rapid point-of-care test for RA.
  • a lateral flow device capable of rapidly providing results as to ACA levels in a biological sample may enable rapid assessment of RA and rheumatic disease.
  • such a lateral flow device may enable quantitation of an amount of ACA in a biological sample of a subject, which in turn may be used to assess severity of disease and/or predict a rate at which rheumatic disease progression may occur.
  • one or more thresholds pertaining to ACA levels (e.g., titer) in a biological sample may be relied upon in order to effectively manage rheumatic disease (e.g., RA).
  • a first threshold, a second threshold, and a third threshold may be used, where ACA levels below the first threshold are used to manage RA in a first manner, where ACA levels greater than the first threshold but below the second threshold are used to manage RA in a second manner, where ACA levels greater than the second threshold but lower than the third threshold are used to manage RA in a third manner, and where ACA levels greater than the third threshold are used to manage RA in a fourth manner.
  • the first manner may be a less aggressive course of action than the second manner, which is in turn is a less aggressive course of action than the third manner, which in turn is a less aggressive course of action than the fourth manner.
  • Lesser aggressive courses of action may include one or more of lesser amounts of therapeutic agents, greater duration between treatments with therapeutic agents, use of lesser numbers of different therapeutic agents, selection of different types of therapeutic agents as compared to more aggressive courses, and less stringent antirheumatic lifestyle modifications.
  • ACA levels below the first threshold may warrant no course of action in terms of managing RA, with increasingly aggressive courses of action increasing upon ACA levels passing the first threshold, the second threshold, or the third threshold.
  • ACA levels discussed herein may range from about 100 U/mL to about 3500 U/mL.
  • the first ACA threshold may comprise about 500 U/mL
  • the second ACA threshold may comprise about 1000 U/mL
  • the third threshold may comprise about 2000 U/mL.
  • Differential management of rheumatic disease may be provided depending on, as mentioned above, whether ACA levels are below the first threshold (e.g., about 500 U/mL or less), greater than the first threshold but lower than the second threshold (e.g., within a range of about 500 U/mL to about 1000 U/mL), greater than the second threshold but lower than the third threshold (e.g., within a range of about 1000 U/mL to about 2000 U/mL), or greater than the third threshold (e.g., greater than about 2000 U/mL).
  • the first threshold e.g., about 500 U/mL or less
  • the second threshold e.g., within a range of about 500 U/mL to about 1000 U/mL
  • the third threshold e.g., within a range of about 1000 U/mL to about 2000 U/mL
  • the third threshold e.g., greater than about 2000 U/mL
  • relative levels of at least two biomarkers with respect to one another may be relied on for determining one or other parameters for management of RA, for example one or more antirheumatic lifestyle modifications.
  • a determination of relative levels of two or more RA biomarkers with respect to one another for effectively managing RA need not necessarily be on a same lateral flow device.
  • a first lateral flow device may be capable of detecting and quantifying ACA, while a second lateral flow device may be capable of detecting and quantifying RF and/or ACPA.
  • quantification may be normalized to control levels as discussed, to enable comparison of relative levels of at least two RA biomarkers between lateral flow test devices.
  • a determination of relative levels of two or more RA biomarkers with respect to one another need not necessarily be confined to just lateral flow assays. Examples of other assays for RF and ACPA have been described herein, and it is within the scope of this disclosure to rely on such assays in order to determine relative levels of at least two biomarkers with respect to each other.
  • a lateral flow device of the present disclosure may be used to determine ACA levels, which in turn may be compared with RF and ACPA levels obtained by other assay formats (e.g., ELISA) not including lateral flow assay formats.
  • ACA point-of-care test was performed by using the lateral flow test device depicted in FIG. 2A.
  • Table 1 indicates that out of 242 subjects with rheumatoid arthritis, 109 subjects were positive with the ACA test (sensitivity of 45% positive) and 112 subjects were positive with the ACCP test (sensitivity of 46% positive). As depicted by Venn diagram 600 shown in FIG. 6, 28 subjects were positive with the ACA test but negative with the ACCP test, and 31 subjects were positive with the ACCP test but negative with the ACA test. As indicated in FIG.
  • the rheumatoid arthritis (RA) autoimmune marker ACA identifies a subset of RA patients not detected by the ACCP assay and increases the sensitivity of detection in combination with the ACCP assay. Furthermore, ACA-positive subjects may be associated with severe/progressive disease because of citrullination of a major serum protein. Thus, the addition of ACA testing to the current standard ACCP testing may increase the rate of detection and indicate potential for more severe disease.

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Abstract

Des modes de réalisation concernent des dispositifs et des procédés d'utilisation pour détecter un ou plusieurs biomarqueurs de maladie rhumatismale chez un sujet. Dans un exemple, un procédé comprend la détection de l'albumine sérique humaine (HSA) citrullinée dans un échantillon biologique par l'obtention de l'échantillon biologique provenant d'un sujet, l'application de l'échantillon biologique à un dispositif à écoulement latéral dans des conditions suffisantes pour la formation d'un immunocomplexe comprenant un anticorps anti-HSA couplé à la HSA citrullinée qui est à son tour couplé à un auto-anticorps anti-HSA citrullinée, la détermination d'une quantité de l'immunocomplexe et la gestion d'une maladie rhumatismale associée au sujet. De cette manière, une maladie rhumatismale peut être efficacement gérée sur la base, au moins en partie, de quantités de HSA citrullinée détectées par une méthodologie de test rapide sur le site de soins.
EP21904515.0A 2020-12-10 2021-12-10 Systèmes et procédés pour détecter des biomarqueurs de la polyarthrite rhumatoïde Pending EP4259818A1 (fr)

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WO2022125972A8 (fr) 2023-07-06

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